The α-Subunit of the Arabidopsis Heterotrimeric G Protein,GPA1, Is a Regulator of Transpiration Efficiency

Land plants must balance CO₂ assimilation with transpiration in order to minimize drought stress and maximize their reproductive success. The ratio of assimilation to transpiration is called transpiration efficiency (TE). TE is under genetic control, although only one specific gene, ERECTA, has been shown to regulate TE. We have found that the α-subunit of the heterotrimeric G protein in Arabidopsis (Arabidopsis thaliana), GPA1, is a regulator of TE. gpa1 mutants, despite having guard cells that are hyposensitive to abscisic acid-induced inhibition of stomatal opening, have increased TE under ample water and drought stress conditions and when treated with exogenous abscisic acid. Leaf-level gas-exchange analysis shows that gpa1 mutants have wild-type assimilation versus internal CO₂ concentration responses but exhibit reduced stomatal conductance compared with ecotype Columbia at ambient and below-ambient internal CO₂ concentrations. The increased TE and reduced whole leaf stomatal conductance of gpa1 can be primarily attributed to stomatal density, which is reduced in gpa1 mutants. GPA1 regulates stomatal density via the control of epidermal cell size and stomata formation. GPA1 promoter::β-glucuronidase lines indicate that the GPA1 promoter is active in the stomatal cell lineage, further supporting a function for GPA1 in stomatal development in true leaves.Land plants, in particular plants that utilize C₃ photosynthesis, must balance CO₂ acquisition with water loss in order to maximize fitness. The water loss cost per unit of biomass acquired can be expressed as transpiration efficiency (TE; also referred to as water-use efficiency), the ratio of CO₂ assimilation (A) to transpiration. TE strongly correlates with the δ¹³C of plant tissue, the ratio of ¹³C to ¹²C relative to a standard (Farquhar et al., 1982, 1989; Dawson et al., 2002). The physiological basis of this correlation is that in plants there is diffusional and biochemical discrimination against ¹³C, the heavier and less abundant stable isotope of carbon. Discrimination against ¹³C decreases with decreasing internal CO₂ concentration (C_i), which can result from either increased A or reduced stomatal conductance (g_s; Farquhar et al., 1982). While it is known that g_s (a main factor controlling transpiration) correlates with A (Wong et al., 1979), genetic variation for TE and/or δ¹³C has been documented in a number of species (Farquhar and Richards, 1984; Virgona et al., 1990; Ehleringer et al., 1991; Comstock and Ehleringer, 1992; Hammer et al., 1997; Lambrides et al., 2004). In Arabidopsis (Arabidopsis thaliana), multiple quantitative trait loci associated with TE have been identified, indicating that TE is under genetic control (Juenger et al., 2005; Masle et al., 2005; McKay et al., 2008). However, only one gene, ERECTA, has been specifically identified as a regulator of TE (Masle et al., 2005). ERECTA encodes a Leu-rich repeat receptor-like kinase (Torii et al., 1996) and regulates TE via the control of stomatal density, g_s, mesophyll cell proliferation, and photosynthetic capacity (Masle et al., 2005).Heterotrimeric G proteins are GTP-binding proteins that function in the transduction of extracellular signals into intracellular responses. In its inactive state, the G protein classically exists as a trimer consisting of an α-subunit (Gα) bound to GDP, a β-subunit (Gβ), and a γ-subunit (Gγ). When a ligand binds to a G protein-coupled receptor (GPCR), a conformational change occurs in the G protein, resulting in the exchange of GDP for GTP and the dissociation of Gα-GTP from the Gβγ dimer. The G protein subunits remain active until the intrinsic GTPase activity of Gα results in the hydrolysis of GTP to GDP and the reassociation of the inactive trimer. The Arabidopsis genome contains canonical Gα and Gβ genes, GPA1 and AGB1, and two genes known to encode Gγs, AGG1 and AGG2 (Assmann, 2002). One likely GPCR, GCR1, has been functionally characterized (Pandey and Assmann, 2004), and additional GPCRs have been predicted using bioinformatics (Moriyama et al., 2006; Gookin et al., 2008) and interaction with GPA1 in yeast-based protein-protein interaction assays (Gookin et al., 2008). Recently, a new class of G proteins, GPCR-type G proteins (GTG1 and GTG2), have been identified in Arabidopsis that also serve as one class of abscisic acid (ABA) receptors (Pandey et al., 2009).Despite the paucity of heterotrimeric G protein subunit genes in the Arabidopsis genome as compared with mammalian systems, functional studies of heterotrimeric G protein mutants suggest that G protein function is diverse in Arabidopsis. G proteins have been shown to function in developmental processes and hormonal and environmental signaling, including stomatal aperture regulation (Perfus-Barbeoch et al., 2004; Joo et al., 2005; Chen et al., 2006; Pandey et al., 2006; Trusov et al., 2006; Warpeha et al., 2007; Fan et al., 2008; Zhang et al., 2008a, 2008b). In response to drought stress, ABA concentration increases in the leaves (Davies and Zhang, 1991; Davies et al., 2005), where it promotes stomatal closure and inhibits stomatal opening (Schroeder et al., 2001). The G protein α- and β-subunit mutants, gpa1 and agb1, respectively, are hyposensitive to ABA inhibition of stomatal opening while displaying wild-type ABA promotion of stomatal closure (Wang et al., 2001; Fan et al., 2008). ABA inhibits stomatal opening in part by inhibiting inward-rectifying K⁺ channels, reducing K⁺ influx and therefore water entry into the cell (Schroeder et al., 2001). ABA inhibition of inward K⁺ channel activity is reduced in both gpa1 and agb1 mutants (Wang et al., 2001; Fan et al., 2008). agg1 and agg2 mutants show no altered regulation of ABA-induced stomatal movements or ion channel activities, suggesting that the genome contains additional unknown Gγ(s) or that heterotrimeric G protein signaling in plants does not always operate according to the mammalian paradigm (Trusov et al., 2008). gcr1 mutants are hypersensitive to both ABA inhibition of opening and ABA promotion of stomatal closure (Pandey et al., 2006). gtg1 gtg2 double mutants show a wild-type response for ABA inhibition of stomatal opening and are hyposensitive in ABA promotion of stomatal closure (Pandey et al., 2009).While the altered stomatal sensitivities of the G protein mutants to ABA suggest that heterotrimeric G proteins may function in the regulation of whole plant water status, few experiments have been performed at the whole leaf or whole plant level. gpa1 mutants in the Wassilewskija background display increased water loss from excised leaves (Wang et al., 2001); however, there are no published reports of experiments assessing whole plant water status in gpa1 or agb1 mutants. gcr1 mutants show reduced water loss from excised leaves, drought tolerance, and improved recovery following the cessation of drought stress (Pandey and Assmann, 2004). In addition to their altered guard cell sensitivities to ABA, gpa1, agb1, and gcr1 mutants are hypersensitive to ABA inhibition of root and seedling development (Pandey et al., 2006), which could have impacts on whole plant water status. Finally, it has been recently reported that gpa1 and agb1 mutants have reduced and increased stomatal densities, respectively, in cotyledons (Zhang et al., 2008a). While stomatal density of leaves can be an important component of whole plant water status, the study by Zhang et al. (2008a) was performed on cotyledons only, whose developmental programs are often independent from those of true leaves (Chandler, 2008). Therefore, it is difficult to infer how this cotyledon phenotype will affect water relations at the whole plant level. Taken together, the stomatal aperture, electrophysiology, and tissue-specific ABA phenotypes of the G protein mutants, in addition to the possibility for altered stomatal density in the G protein mutant leaves, make it difficult to predict how G proteins contribute to the regulation of whole-plant TE. For example, the ABA-hyposensitive stomatal phenotype of gpa1 could result in increased transpiration, possibly reducing TE under certain conditions. Conversely, if gpa1 mutant leaves have reduced stomatal density, transpiration may be reduced, which could enhance TE under a range of conditions. Previous attempts to address the contributions of G proteins to whole plant transpiration, TE, and drought response using excised leaf/rosette assays to measure water loss are not sufficient, because both transpiration and A must be taken into account. Therefore, we investigated the role of GPA1 in regulating TE under ample water and drought stress conditions and in the presence of ABA. We have identified GPA1 as a negative regulator of TE in Arabidopsis via the control of g_s and stomatal proliferation.     

Protection of Armadillo/β-Catenin by Armless,a Novel Positive Regulator of Wingless Signaling

The Wingless (Wg/Wnt) signaling pathway is essential for metazoan development, where it is central to tissue growth and cellular differentiation. Deregulated Wg pathway activation underlies severe developmental abnormalities, as well as carcinogenesis. Armadillo/β-Catenin plays a key role in the Wg transduction cascade; its cytoplasmic and nuclear levels directly determine the output activity of Wg signaling and are thus tightly controlled. In all current models, once Arm is targeted for degradation by the Arm/β-Catenin destruction complex, its fate is viewed as set. We identified a novel Wg/Wnt pathway component, Armless (Als), which is required for Wg target gene expression in a cell-autonomous manner. We found by genetic and biochemical analyses that Als functions downstream of the destruction complex, at the level of the SCF/Slimb/βTRCP E3 Ub ligase. In the absence of Als, Arm levels are severely reduced. We show by biochemical and in vivo studies that Als interacts directly with Ter94, an AAA ATPase known to associate with E3 ligases and to drive protein turnover. We suggest that Als antagonizes Ter94''s positive effect on E3 ligase function and propose that Als promotes Wg signaling by rescuing Arm from proteolytic degradation, spotlighting an unexpected step where the Wg pathway signal is modulated.     

Calcium: Just Another Regulator in the Machinery of Life?

*BACKGROUND: Current hypotheses imply that stimulus-response systems in plants are networks of signal transduction pathways. It is usually assumed that these pathways connect receptors with effectors via chains of molecular events. Diverse intermediate signalling components (transducers) participate in these processes. However, many cellular transducers respond to several stimuli. Hence, there are no discrete chains but rather pathways that interconnect network-modules of different command structure. In particular, the cytosolic free Ca2+ concentration ([Ca2+](cyt)) is thought to perform many different tasks in a wide range of cellular events. However, this range of putative functions is so wide that it is often questioned how Ca2+ can comply with the definition of a second messenger. *THE Ca2+ SIGNATURE HYPOTHESIS: Some authors have suggested the concept of a specific signature of the ([Ca2+](cyt)) response. This implies that characteristics of the time course of changes in ([Ca2+](cyt)) and their localized sites of appearance in cells are used by the plant to identify the type and intensity of the stimulus. This hypothesis has triggered many investigations, which have yielded contradictory results. * THE CURRENT PICTURE: Much evidence suggests that the functions of calcium can be grouped into three classes: Ca2+ as a protective agent, Ca2+ as a chemical switch and Ca2+ as a 'digital' information carrier. Examples of the first two classes are presented here. The third is more controversial; while some investigations seem to support this idea, others call the Ca2+ signature hypothesis into question. Further investigations are needed to shed more light on Ca(2+)-driven signalling cascades.     

SPSB1, a Novel Negative Regulator of the Transforming Growth Factor-β Signaling Pathway Targeting the Type II Receptor

Appropriate cellular signaling is essential to control cell proliferation, differentiation, and cell death. Aberrant signaling can have devastating consequences and lead to disease states, including cancer. The transforming growth factor-β (TGF-β) signaling pathway is a prominent signaling pathway that has been tightly regulated in normal cells, whereas its deregulation strongly correlates with the progression of human cancers. The regulation of the TGF-β signaling pathway involves a variety of physiological regulators. Many of these molecules act to alter the activity of Smad proteins. In contrast, the number of molecules known to affect the TGF-β signaling pathway at the receptor level is relatively low, and there are no known direct modulators for the TGF-β type II receptor (TβRII). Here we identify SPSB1 (a Spry domain-containing Socs box protein) as a novel regulator of the TGF-β signaling pathway. SPSB1 negatively regulates the TGF-β signaling pathway through its interaction with both endogenous and overexpressed TβRII (and not TβRI) via its Spry domain. As such, TβRII and SPSB1 co-localize on the cell membrane. SPSB1 maintains TβRII at a low level by enhancing the ubiquitination levels and degradation rates of TβRII through its Socs box. More importantly, silencing SPSB1 by siRNA results in enhanced TGF-β signaling and migration and invasion of tumor cells.     

Structural and Functional Analysis of the Regulator of G Protein Signaling 2-Gαq Complex

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Highlights? Two structures of the RGS2-Gα_q complex were determined by X-ray crystallography ? RGS2 binds Gα_q in a manner distinct from how other RGS proteins bind Gα_i/o ? In its distinct pose, RGS2 forms extensive contacts with the α-helical domain of Gα_q ? Helical domain contacts contribute to binding affinity and GAP potency of RGS2     

Differential Regulation of Carbon Partitioning by the Central Growth Regulator Target of Rapamycin （TOR）

Target of rapamycin （TOR） is an evolutionary conserved ser- ine/threonine protein kinase found in yeast, plants, and ani- mals. TOR plays a central role in sensing nutrients and energy status, growth factors, and other environmental signals, and integrates these cues to synchronize cell growth-related pro- cesses （Laplante and Sabatini, 2012）. In yeast and animal cells, TOR acts as the catalytic component in two structurally and functionally distinct protein complexes termed TORC （TOR complex） 1 and TORC2. TOR seems to be localized specifi- cally to the endomembrane system and the nucleus with a certain mobility, depending on the environmental conditions （Laplante and Sabatini, 2012）. The three major components of TORCl, namely TOR, RAPTOR （Regulatory associated protein of mTOR）, and LST8 （Lethal with Sec13 protein 8）, are present in the genome of Arabidopsis, and interactions between TOR and RAPTOR, as well as TOR and LST8, occur also in plants, indicating the existence of a plant TORC1 （Moreau et al., 2012）. Here, we highlight the possible role of TOR in carbon utilization and growth regulation in Arabidopsis.     

Circulating Levels of Human salusin-β,a Potent Hemodynamic and Atherogenesis Regulator

Using bioinformatics analysis, we previously identified salusin-β, an endogenous bioactive peptide with diverse physiological activities. Salusin-β is abundantly expressed in the neuroendocrine system and in systemic endocrine cells/macrophages. Salusin-β acutely regulates hemodynamics and chronically induces atherosclerosis, but its unique physicochemical characteristics to tightly adhere to all types of plastic and glassware have prevented elucidation of its precise pathophysiological role. To quantitate plasma total salusin-β concentrations, we produced rabbit and chicken polyclonal antibodies against the C- and N-terminal end sequences, circumvented its sticky nature, and successfully established a sandwich enzyme-linked immunosorbent assay (ELISA). Salusin-β was abundantly present in the plasma of healthy volunteers, ranging from 1.9 to 6.6 nmol/L. Reverse phase-high performance liquid chromatography analysis showed that a single immunoreactive salusin-β peak coincided with synthetic authentic salusin-β. Plasma salusin-β concentrations were unaffected by postural changes and by potent vasopressin release stimuli, such as hypertonic saline infusion or smoking. However, salusin-β concentrations showed significant circadian variation; concentrations were high during the daytime and reached the lowest concentrations in the early morning. Plasma salusin-β levels in subjects with diabetes mellitus, coronary artery disease, and cerebrovascular disease showed distinctly higher levels than healthy controls. Patients with panhypopituitarism combined with complete central diabetes insipidus also showed significantly higher plasma salusin-β levels. Therefore, the ELISA system developed in this study will be useful for evaluating circulating total salusin-β levels and for confirming the presence of authentic salusin-β in human plasma. The obtained results suggest a limited contribution of the neuroendocrine system to peripheral total salusin-β concentrations and a role for plasma total salusin-β concentrations as an indicator of systemic vascular diseases.     

Evidence Against the “Y–T Coupling” Mechanism of Activation in the Response Regulator NtrC

The dominant theory on the mechanism of response regulators activation in two-component bacterial signaling systems is the “Y–T coupling” mechanism, wherein the χ₁ rotameric state of a highly conserved aromatic residue correlates with the activation of the protein via structural rearrangements coupled to a conserved tyrosine. In this paper, we present evidence that, in the receiver domain of the response regulator nitrogen regulatory protein C (NtrC^R), the interconversion of this tyrosine (Y101) between its rotameric states is actually faster than the rate of inactive/active conversion and is not correlated to the activation process. Data gathered from NMR relaxation dispersion experiments show that a subset of residues surrounding the conserved tyrosine sense a process that is occurring at a faster rate than the inactive/active conformational transition. We show that this process is related to χ₁ rotamer exchange of Y101 and that mutation of this aromatic residue to a leucine eliminated this second faster process without affecting activation. Computational simulations of NtrC^R in its active conformation further demonstrate that the rotameric state of Y101 is uncorrelated with the global conformational transition during activation. Moreover, the tyrosine does not appear to be involved in the stabilization of the active form upon phosphorylation and is not essential in propagating the signal downstream for ATPase activity of the central domain. Our data provide experimental evidence against the generally accepted “Y–T coupling” mechanism of activation in NtrC^R.     

α-Glucosidase at the tip of the contact chemosensory seta of the blowfly, Phormia regina

α-Glucosidase activity was detected at the tip of the labellar contact chemosensory hair of the blowfly, Phormia regina. The enzyme split about 1 pmole of sucrose per hr per hair on average and the Michaelis constant for sucrose was about 50 mM. The activity of the enzyme was not solubilized into the incubation solution, but stuck stably to the tip of the sensory hair. From the cut end of the sensory hair a high activity of α-glucosidase eluted out. But its Michaelis constant was smaller by far than the one at the tip, suggesting that different types of α-glucosidase isozymes exist in the hair. The possibility that the enzyme at the tip of the sensory hair could be the sugar receptor is discussed.